1. Field of the Invention
The present invention relates to the production and use of soluble analogs of thrombomodulin that retain activity after exposure to oxidants. These analogs are manufactured using recombinant DNA technology and are useful in, for example, antithrombotic therapy. Novel proteins, nucleic acid gene sequences, vectors, pharmaceuticals and methods of inhibiting thrombotic activity are disclosed.
2. Information Disclosure
There are many disease states that would benefit from treatment with a safe and effective anticoagulant/antithrombotic. The nature of these conditions varies. For example, anticoagulant therapy is useful in acute conditions such as during thrombolytic therapy in myocardial infarction or in treatment of disseminated intravascular coagulation (DIC) associated with, for example, septicemia. Anticoagulants are also useful for less acute conditions, such as chronic use in patients that have received heart valve implants or prophylactic use in surgery patients to reduce the risk of deep venous thrombosis (DVT). The anticoagulants currently approved for use in humans are not uniformly effective and a need exists for more efficacious compounds (See, for example, Prevention of Venous Thrombosis and Pulmonary Embolism, Consensus Development Conference Statement, NIH, 1986, 6(2):1-23).
Thrombomodulin is a membrane protein that has demonstrated anticoagulant properties. In humans, it is widely distributed on the endothelium of the vasculature and lymphatics except in the central nervous system. It functions as a receptor for thrombin, a central enzyme in the coagulation cascade. When free, thrombin promotes coagulation both directly by converting fibrinogen to fibrin and activating platelets, and indirectly through activation of other proteins in the coagulation cascade (Factors V, VIII and XIII, for example). When bound to thrombomodulin, however, the procoagulant activities of thrombin are inhibited, and its chief function is switched to the activation of protein C. Activated protein C in turn disrupts the coagulation process at several points. (See, for example, N Esmon, et al, (1982) J. Biol. Chem. 257:859-864, H. Salem, et al, (1983) J. Biol. Chem. 259:12246-12251).
The gene encoding native thrombomodulin has been isolated and sequenced from several species, both in its genomic form and as a cDNA (R. Jackman, et al, (1986) PNAS 83:8834-8838 and (1987) 84:6425-6429, both of which are herein incorporated by reference). Comparisons with known proteins, such as the LDL receptor, have suggested functional domains (D. Wen, et al, (1987) Biochemistry 26:4350-4357). One study has suggested that the fifth and sixth epidermal growth factor (EGF)-like domains have the capacity to bind thrombin (S. Kurosawa, et al, (1988) J. Biol. Chem. 263:5993-5996; another suggests that EGF-like domains 4, 5, and 6 are sufficient to act as a cofactor for thrombin mediated protein C activating activity. (Zushi, et al, (1989) J. Biol. Chem. 264:10351-10353).
Thrombomodulin in its natural form is not suitable for anticoagulant therapy as it is membrane-bound, due to its inherent amino acid sequence, and is insoluble without detergent treatment. It is present in such small amounts (about 300 mg thrombomodulin/person) that purification from autopsy or biopsy samples is impractical.
The inventors have also discovered that native thrombomodulin is susceptible to oxidation and when oxidized loses its ability to promote the activation of protein C. Many of the disease conditions requiring anticoagulation are also associated with high levels of toxic oxygen radicals, which can inactivate biomolecules and cause significant tissue damage. Examples of these conditions are reperfusion injury associated with myocardial infarction, DIC associated with septicemia, and alveolar fibrosis associated with adult respiratory distress syndrome. (See, Otani, H., et al, (1984) Circ. Res. 55:168-175, Saldeen, T., (1983) Surg. Clin. N.A. 63(2):285-304, and Idell, S., et al, (1989) J. Clin. Inv. 84:695-705.) In addition, any wound, such as occurring in surgical procedures, involves the influx of activated monocytes, polymorphonuclear leukocytes, etc. which can create toxic oxygen species as well as releasing a host of proteolytic enzymes, such as elastase. The connection between endothelial cell damage, inflammation and thrombosis has long been recognized (See The Molecular and Cellular Biology of Wound Repair. ed. Clark, R.A.F. and P.M. Henson 1988, for example). However, the inventors are the first to recognize that thrombomodulin is subject to inactivation by exposure to toxic oxygen species and that this likely plays a significant role in many pathogenic states.
Soluble thrombomodulin-like molecules have been detected at very low amounts in human plasma and urine. These molecules have a reduced ability to promote protein C activation, and it is possible that they have been rendered at least partially inactive, due at least in part to oxidation. It has been suggested that these molecules are degradation products of the membrane bound molecule (H. Ishii and P. Majerus, (1985) J. Clin. Inv. 76:2178-2181), but they are present in such low amounts that they have been difficult to characterize (.about.0.8 mg/adult male). Proteolytic fragments of the purified native molecule have been produced using trypsin or elastase. (See, Ishii, supra, Kurosawa, et al, (1988) J. Biol. Chem. 263:5593-5996 and Stearns, et al, (1989) J. Biol. Chem. 264:3352-3356). Some of these fragments retain the ability to promote thrombin mediated activation of protein C in vitro.
Soluble analogs of thrombomodulin that retain most, if not all of, the activities of the native protein have been produced and are described in copending, coassigned applications U.S. Ser. No. 312,141 filed Feb. 17, 1989, U.S. Ser. No. 345,372 filed Apr. 28, 1989, U.S. Ser. No. 406,941 filed Sep. 13, 1989 and WO 90/00955 filed Feb. 16, 1990 and are herein incorporated by reference. Additional references include EP 290,419 and WO 88/05053, which discloses cDNA encoding the human thrombomodulin protein. Analogs of thrombomodulin have also been described in WO 88/05053, which discloses analogs with varying numbers of EGF-like domains.
There is a need for new compositions that exhibit the anticoagulant properties of thrombomodulin, are soluble in plasma, are resistant to inactivation by exposure to oxidants, and are easily produced in large quantities. The present invention fulfills these and other needs.